It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. It is the generally object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides to Watson-Crick base pairs of RNA or single stranded DNA. Such base pairs are said to be complementary to one another.
Applications of oligonucleotides as diagnostics, research reagents, and potential therapeutic agents requires that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes, or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend partly on the initial stability of oligonucleotides towards nuclease degradation. Further, these functions depend on specificity of the oligonucleotide or oligonucleotide analog for a target molecule.
A serious deficiency of naturally occuring oligonucleotides and existing oligonucleotide analogs for these purposes, particularly those for antisense therapeutics, is the enzymatic degradation of the admininstered oligonucleotide by a variety of ubiquitous nucleolytic enzymes, intracellularly and extracellularly located, hereinafter refered to as "nucleases". It is unlikely that unmodified oligonucleotides will be useful thereapeutic agents because they are rapidly degraded by nucleases. Modification of oligonucleotides to render them resistant to nucleases is therefore greatly desired.
Modifications of oligonucleotides to enhance nuclease resistance has generally taken place on the sugar-phosphate backbone, particularly on the phosphorous atom. Phosphorothioates have been reported to exhibit resistance to nucleases. In addition, phosphorothioate oligonucleotides are generally more chemically stable than natural phosphodiester oligonucleotides. Phosphorothioate oligonucleotides also exhibit solubility in aqueous media. Further, phosphorothioate oligonucleotide-RNA heteroduplexes can serve as substrates for endogenous RNase H. Additionally, phosphorothioate oligonucleotides exhibit high thermodynamic stability. However, while the ability of an antisense oligonucleotide to bind to specific DNA or RNA with fidelity is fundamental to antisense methodology, modifications at the phosphorous oligonucleotides, while exhibiting various degrees of nuclease resistance, have generally suffered from inferior hybridization properties. Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of Gene Expression (CRC Press, Inc., Boca Raton, Fla. 1989).
One reason for this inferior hybridization may be due to the prochiral nature of the phosphorous atom, modifications on the internal phosphorous atom of modified phosphorous oligonucleotides results in Rp and Sp stereoisomers. Since a practical synthesis of oligonucleotides having substantially all-Rp or all-Sp phosphate intersugar linkages has been unknown, oligonucleotides with modifications at the phosphorous atoms, wherein the resulting molecule has nonsymmetrical substituents, are racemic mixtures having 2.sup.n isomers with n equal to the number of phosphorothioate intersugar linkages in the oligonucleotide. Thus a 15-mer phosphorothioate oligonucleotide, containing 14 asymmetric centers has 2.sup.14 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to hybridize to a target mRNA or DNA with sufficient affinity to be useful in antisense or probe technology.
Attempts to study chemically synthesized phosphorothioate oligonucleotides having chirally pure intersugar linkages has been limited to molecules having only one or two diastereomeric intersugar linkages. The effects of induced chirality in chemically synthesized racemic mixtures of sequence specific phosphorothioate oligonucleotides has not been assessed since synthesis of oligonucleotides having chirally pure intersugar linkages has yet to be accomplished by automated synthesis. This is due to the non-stereospecific incorporation of sulfur during automated synthesis. For example, Stec, W. J., Zon, G. and Uznanski, B., J. Chromatography, 326:263 (1985), synthesized certain oligonucleotide phosphorothioates having racemic intersugar linkages; however, they were able to resolve only the diastereomers of certain small oligomers having one or, at most, two diastereomeric phosphorous intersugar linkages.
While chemical synthesis of oligonucleotides having chirally pure intersugar linkages has been limited to either small dimers and trimers or to homopolymers or to, at best, two diastereomeric phosphorous intersugar linkages, the synthesis of phosphorothioates having all-Rp intersugar linkages using enzymatic methods has been investigated by several authors. Burgers, P. M. J. and Eckstein, F., J. Biological Chemistry, 254:6889 (1979), and Gupta, A., DeBrosse, C., and Benkovic, S. J. J. Bio. Chem., 256:7689 (1982) enzymatically synthesized diastereomeric polydeoxy-adenylic acid having phosphorothioate intersugar linkages. Brody, R. S. and Frey, P. S., Biochemistry, 20:1245 (1981); Eckstein, F. and Jovin, T. M., Biochemistry, 2:4546 (1983); Brody, R. S., Adler, S., Modrich, P., Stec, W. J., Leznikowski, Z. J., and Frey, P. A., Biochemistry, 21:2570-2572 (1982); and Romaniuk, P. J. and Eckstein, F., J. Biol. Chem., 257:7684-7688 (1982) all enzymatically synthesized poly TpA and poly ApT phosphorothioates while Burgers, P. M. J. and Eckstein, F. Proc. Natl. Acad. Sci. USA, 75:4798-4800 (1978) enzymatically synthesized poly UpA phosphorothioates. Cruse, W. B. T., Salisbury, T., Brown, T., Cosstick, R. Eckstein, F. and Kennard, O., J. Mol. Biol., 192:891 (1986), linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer.
Studies of homopolymeric oligonucleotides which have chirally pure intersugar linkages have indicated that in some cases oligonucleotides having substantially all-Rp intersugar linkages may exhibit greater binding fidelity to target molecules.
The relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T.sub.m), a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coil (unhybridized) forms are present. T.sub.m is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T.sub.m. The higher the T.sub.m, the greater the strength of the binding of the strands. Non-Watson-Crick base pairing has a strong destabilizing effect on the T.sub.m.
In a preliminary report, see Stec, J. W., Oligonucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications. Meeting abstracts, Jun. 18-21, 1989 thymidine homopolymer octamers having all but one linkage being modified phosphate linkages ("all except one") Rp stereoconfiguration or "all except one" Sp stereoconfiguration in the intersugar linkages were formed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester bond. It was noted that a Rp "all except one" methylphosphonate non-sequence specific thymidine homopolymer octomer, i.e. a (dT).sub.8 mer having all but one Rp intersugar linkage, formed a thermodynamically more stable hybrid (Tm 38.degree. C.) with 15 mer deoxyadenosine homopolymer, i.e. a d(A).sub.15 mer, than a hybrid formed by a similar thymidine homopolymer having all-except-one Sp configuration methylphosphonate linkages and of d(A).sub.15 mer (Tm &lt;0.degree. C.), i.e. a d(T).sub.15 having all but one Sp intersugar linkage. A hybrid between a (dT).sub.8 mer having natural phosphodiester linkages, i.e. octathymidylic acid, and a d(A) l.sub.5 mer was reported to have a Tm of 14.degree. C.
More recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R. and Watanabe, K., Nucleic Acids Research, 19:547 (1991), enzymatically synthesized mRNAs intermittently incorporating Rp diastereomeric phosphorothioate linkages for use in translation systems. Ueda, et al. employed T7 coliphane DNA having seventeen promoters and one termination site for T7 RNA polymerase. In vitro synthesis by T7 RNA polymerase produced mRNAs having from several hundred to tens of thousands of nucleotides.
Backbone chirality may also effect the susceptibility of a phosphorothioate oligonucleotide-RNA heteroduplex to serve as a substrate for RNase H activity. The ability to serve as a template for RNAse H will likely have considerable therapeutic importance since it has been suggested that RNAse H may cause a terminating event by cleavage of the RNA component in an RNA-oligonucleotide heteroduplex. With oligonucleotides containing racemic mixtures of Rp and Sp intersugar linkages, it is not known if all phosphorothioate oligonucleotides can function equally as substrates for RNase H. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation by "hybridization arrest". Thus, increasing the percentage of phosphorothioate oligonucleotides that can serve as substrates for RNAse H in a heteroduplex will likely lead to a more efficacious compound for antisense therapy.
To enhance binding fidelty and thus antisense methodology, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired. Further, such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages would lead to more efficacious compounds for antisense therapy. However, until now little success has been achieved in an effort to synthesize such molecules. Therefore, simple methods of synthesizing phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired.